CasOligo
CasOligo is a R package to search the 20nt gRNA-target-site oligonucleotide sequence within 18S rRNA gene for designing the taxon-specific gRNA used for CRISPR-Cas Selective Amplicon Sequencing (CCSAS, Zhong et al., 2020) to assess the eukaryotic microbiome of hosts (e.g. metazoans, plant). Taxon-specific gRNA would guide the Cas nuclease to cut 18S rRNA gene of desired hosts specifically, but not of protists and fungi. This results in a sequencing-library highly enriched in 18S amplicons from protists and fungi, allowing for high-resolution surveys of the taxonomic composition and structure of the eukaryotic microbes associated with the host. CCSAS provides a new way to obtain high-resolution taxonomic data for the eukaryotic microbiomes of plants, animals and other metazoa.
To facilitate application of CCSAS, we designed gRNA-taxon-sites and gRNAs for almost all metazoan and metaphyta taxa that are currently available at SILVA (Quast et al., 2003), creating a gRNA-taxon-sites database for researchers who want to apply to their own organisms for various purposes. Beyond that, the CasOligo package provides an oligonucleotide design function, Cas9.gRNA.oligo2 function, that can be used to design custom gRNA for any gene for which the sequence is known and there is a reference database, including genes encoding other regions of 18S rRNA (e.g. 16S, 23S or ITS), or metabolic genes (e.g. COX1). Thus, CCSAS makes it possible to study the genetic diversity of any gene in complex systems, including those that are rare, by removing any sequence that would otherwise dominate the data. The sequence-specific removal of any amplicon has a wide range of applications, including pathogen diagnosis, and studies of symbiosis and microbiome therapy.
